Assessment of Vibrionaceae prevalence in seafood from Qidong market and analysis of Vibrio parahaemolyticus strains

The aim of this study was to investigate the prevalence of Vibrionaceae family in retail seafood products available in the Qidong market during the summer of 2023 and to characterize Vibrio parahaemolyticus isolates, given that this bacterium is the leading cause of seafood-associated food poisoning. We successfully isolated a total of 240 Vibrionaceae strains from a pool of 718 seafood samples. The breakdown of the isolates included 146 Photobacterium damselae, 59 V. parahaemolyticus, 18 V. campbellii, and 11 V. alginolyticus. Among these, P. damselae and V. parahaemolyticus were the predominant species, with respective prevalence rates of 20.3% and 8.2%. Interestingly, all 59 isolates of V. parahaemolyticus were identified as non-pathogenic. They demonstrated proficiency in swimming and swarming motility and were capable of forming biofilms across a range of temperatures. In terms of antibiotic resistance, the V. parahaemolyticus isolates showed high resistance to ampicillin, intermediate resistance to cefuroxime and cefazolin, and were sensitive to the other antibiotics evaluated. The findings of this study may offer valuable insights and theoretical support for enhancing seafood safety measures in Qidong City.


Introduction
The family Vibrionaceae, a member of the sub-phylum Gammaproteobacteria, includes genera such as Enterovibrio, Grimontia, Photobacterium, and Vibrio [1].They are cosmopolitan, being distributed widely in marine environments, including seawater, sediments, and among marine flora and fauna [1].The Vibrio genus encompasses over 100 species, with approximately 12 of these species known to be pathogenic to humans.The most prevalent among these are V. cholerae, V. parahaemolyticus and V. vulnificus [2,3].Typically, human infections from Vibrio species arise from the consumption of contaminated water or the ingestion of raw and undercooked contaminated seafood [2].The number of Vibrio cells increases with the rise of seawater temperature, and the threat of these bacteria pose to human health seems to be on the rise with global warming [2,4].
In China, a variety of antimicrobial agents, including tetracycline, ampicillin, sulfonamides, and gentamicin, are permitted for managing bacterial infections in aquaculture.However, the improper use of antibiotics has resulted in bacteria developing resistance to multiple antibiotics.Studies have reported that certain Vibrio species, including V. parahaemolyticus, exhibit resistance to a range of antibiotics [15].Genes that confer resistance to multiple antibiotics such as ampicillin, ceftazidime, gentamicin, chloramphenicol, and kanamycin have been identified within the genomes of V. parahaemolyticus isolates [16,17].Moreover, the ability of nearly all V. parahaemolyticus isolates to form robust biofilms on the surfaces of seafood products is noteworthy [9,18].Biofilms, which are communities of microorganisms encased in an extracellular matrix, provide the bacteria within them with enhanced resilience to harsh conditions [18,19].The formation of these biofilms is facilitated by various structures, including flagella, pili, adhesion proteins, and exopolysaccharides [19].
The Lvsi Fishing Ground, one of the China's four major fishing grounds, covers an area of approximately 30,000 square kilometers and is located in Qidong, Nantong, Jiangsu Province, bordering the Yellow Sea.This region is a vital hub for marine life, offering a rich feeding and breeding environment for a variety of fish, shrimp, shellfish, crab, and other economically significant species.The presence and concentration of pathogenic Vibrionaceae in seafood are crucial factors impacting food safety.While numerous studies have examined the genetic, pathogenic, and phenotypic traits of significant pathogenic strains of the Vibrionaceae isolated in Jiangsu Province, they have predominantly concentrated on individual species, particularly V. parahaemolyticus [9,[20][21][22].These studies have not, however, explored the prevalence of strains within the entire Vibrionaceae family in seafood.In this study, our aim was to assess the incidence of Vibrionaceae in retail seafood products available in the Qidong market during the summer of 2023.We focused on the characterization of virulence genes, biofilm formation capabilities, flagella-driven motility, and antimicrobial resistance among V. parahaemolyticus isolates obtained from these seafood samples.

Sample collection
In the routine process of transporting fish to various markets, it's common practice to utilize liquid nitrogen for rapid freezing or ice blocks for cooling to preserve the freshness and quality of the fish.Fresh seafood was collected from the seafood stalls at the retail market in Huilong Town, Qidong City, from June to November of 2023.This collection period was chosen because there is a linear correlation observed between the water temperature and the population density of Vibrionaceae family members [23,24].A total of 718 samples were collected in this study, including 30 commonly consumed seafood products.These encompassed 16 species of fish, 7 species of shrimp, 3 species of shellfish, 2 species of crab, and 2 species of cephalopods (Table 1).Most of the fish samples were collected from the viscera, gills, or heads.Tissue samples from the same individuals were placed in a sterile sampling bag.Some smaller fish were placed directly in the bag and sent to Department of Clinical Laboratory of Nantong Third People's Hospital.Shellfish were individually bagged and, upon arrival at the laboratory,  their shells were brushed and opened to extract all the contents.Shrimp were transported to the laboratory with only their heads taken for analysis, while crabs were either taken with their gills or with their detached legs.All samples must arrive at the laboratory within two hours and should be processed within half an hour of arrival.

Isolation of bacterial strains belonging to Vibrionaceae
Bacterial strains belonging to Vibrionaceae family were similarly isolated as previously described [9].Briefly, each sample (10 g) was homogenized in 5 ml of sterile phosphate-buffered saline (PBS, pH 7.4) using a lab blender.The homogenate was diluted 1: 50 into 5 ml Alkaline Peptone Water (APW) (Polypeptone 10 g/l; Sodium chloride 30 g/l; pH8.6) and incubated at 30˚C with shaking for 6 h.The APW-enriched culture was diluted 1, 000-fold with PBS, and then 200 μl of the diluted sample were spread on thiosulphate citrate bile salts sucrose (TCBS; Beijing Land Bridge, China) agar plate, and incubated at 37˚C for 24 h.Suspected colonies were selected and characterized by the VITEK MS system (bioMerieux, France) with the platform v3.2 according to the manufacturer's instructions.From the same sample, among clones with the same colony morphology, only one strain was randomly selected.In addition, ethics approval was not requested because no human and experimental animal subjects were involved.

Polymerase chain reaction (PCR) assay
V. parahaemolyticus genomic DNA was extracted similarly as previously study [9].Briefly, 15 μl glycerol-preserved bacterial strain was inoculated into 5 ml 2.5% Bacto heart infusion (HI; BD Bioscience, USA) broth and incubated at 37˚C with shaking for 12 h, followed by centrifugation at 5000 g for 3 min.The genomic DNA was extracted by a QIAamp DNA mini Kit (Qiagen, Germany), and the concentration of DNA solution was measured by a NanoDrop spectrophotometry (ThermoFisher Scientific, USA).PCR was used to detect the presence of the following genetic markers: tlh, tdh, trh, toxR, toxR/new, PGS, orf8, HU-a, ure, Mtase, vopC (VPA1321), VPA1376, vopQ (VP1680), VP1409, and vipA2 (VPA1035).PCR amplification was conducted under the following conditions [9]: an initial pre-denaturation step at 95˚C for 5 min, succeeded by 30 cycles consisting of denaturation at 94˚C for 50 s, annealing at 54˚C for 50 s, and extension at 72˚C for 50 s.A final extension step was performed at 72˚C for 5 min.The PCR products were subsequently visualized using 1% agarose gel electrophoresis.Primers used in this work are listed in Table 2.

Crystal violet (CV) staining
The CV staining assay was performed as previously described [25].Briefly, the overnight bacterial cell culture was diluted 50-fold into 5 ml HI broth and cultured at 37˚C with shaking 200 rpm to an OD 600 value of 1.4.The resultant culture was 50-fold diluted into 2 ml Difco marine (M) broth 2216 (BD Biosciences, USA) in a 96-well plate (Corning Inc., USA) and then grew at 4, 25 or 37˚C with shaking at 150 rpm for 24 h.Biofilm cells were stained with 0.1% CV, which was dissolved with 20% acetic acid.The OD 570 values of acetic acid solutions were measured as the index of CV staining.Biofilms were categorized into three grades based on their OD 570 values: weak (OD 570 < 0.2), moderate (0.2 � OD 570 < 0.7), and strong (OD 570 � 0.7) [9].

Motility-associated assays
The swimming and swarming motility were assessed as previously described [26].Briefly, the overnight cell cultures were diluted 50-fold into 5 ml HI broth and cultured at 37˚C with shaking at 200 rpm to an OD 600 value of 1.

Replicates and statistical methods
PCR and AST were performed three times with the similar results.The CV staining assay, swimming and swarming motility were performed at least three independent bacterial cultures with three replicates for each, and the results were expressed as the mean ± standard deviation (SD).Paired Student's t-tests and two-way ANOVA with Tukey's post hoc corrections for multiple comparisons were used to calculate statistical significance, with P < 0.01 considered significant.

Virulence gene profiles in V. parahaemolyticus isolates
V. parahaemolyticus is the leading cause of seafood-associated gastroenteritis in coastal areas [27,28].Therefore, in this study, the virulence gene profiles in the 59 V. parahaemolyticus isolates were examined.As detailed in

Biofilm formation by the V. parahaemolyticus isolates
The CV staining assay was employed to assess the biofilm-forming capacities of the 59 V. parahaemolyticus isolates.As depicted in Fig 1A, all isolates demonstrated the ability to form biofilms.At 4˚C, the majority were classified as weak biofilm producers, with only two isolates exhibiting moderate biofilm formation (Fig 1A and 1B).In contrast, at 25˚C and 37˚C, the majority of isolates were identified as moderate or strong biofilm producers, with a notable Table 3. Isolates and detection rates of Vibrio species from seafood samples.

Swimming and swarming motility of the V. parahaemolyticus isolates
V. parahaemolyticus is capable of swimming in liquids and swarming over surfaces or in viscous conditions [29].In this study, we investigated the swimming and swarming motility of each isolate, assessing the levels of these two types of motility using a previously described method [9] and 2D).These findings align with previous observations that the majority of V. parahaemolyticus isolates possess notably strong motility [9,30].

Antibiotics resistance profiles of the V. parahaemolyticus isolates
AST was performed on 59 V. parahaemolyticus isolates using a panel of 14 antibiotics.As shown in Table 5, sensitivity to ampicillin was observed in only 8.5% of the isolates, with a significant majority (83.0%) exhibiting resistance.A high level of intermediate resistance was noted for cefuroxime (93.2%) and cefazolin (84.7%).Conversely, all isolates displayed sensitivity to the remaining 11 antibiotics listed in Table 5.

Discussion
In this study, a total of 240 bacterial isolates belonging to Vibrionaceae family were recovered from 718 seafood samples collected from the retail seafood products market in Qidong city from June to November of 2023.Of these, 146 were identified as P. damselae, 59 as V. parahaemolyticus, 18 as V. campbellii, 11 as V. alginolyticus, and 6 as other Vibrio species (Table 3).The isolation rates were 20.3% for P. damselae, 8.2% for V. parahaemolyticus, 2.5% for V. campbellii, and less than 2.0% for the remaining Vibrionaceae family (Table 3).P. damselae, a significant pathogen in marine ecosystems, is known to cause infections in various marine animals such as fish, molluscs, crustaceans, and even cetaceans [31].It poses a risk to human health as an opportunistic pathogen, potentially leading to fatal outcomes through the consumption of raw seafood or exposure to seawater [31].Therefore, it is imperative that the https://doi.org/10.1371/journal.pone.0309304.t004 safety concerns associated with P. damselae in seafood are addressed with due diligence.Additionally, extensive literature exists that examines the prevalence of V. parahaemolyticus in seafood products [20,21,27,28,[32][33][34][35]; however, the detection rates reported vary significantly across studies.For instance, studies have reported detection rates of 28.8% in seafood from ''La Nueva Viga" market in Mexico City [36], 40.3% from 7 stations along the Korean coast [37], 28.57% in the Greater Sacramento area in California [34], and 21.7% in local seafood market of Northern Thailand [33].While the isolation rate of V. parahaemolyticus observed in our study (8.2%) was significantly lower than rates reported in some existing literature, the underlying reasons for this discrepancy remain unclear.
V. parahaemolyticus is the primary causative agent of seafood-associated food poisoning.The presence of tdh and trh genes serves as molecular indicators of pathogenicity in V. parahaemolyticus isolates [5].However, the majority of V. parahaemolyticus found in the environment are non-pathogenic and typically lack the tdh and/or trh genes [30,35,38,39].It is noteworthy that all V. parahaemolyticus strains examined in this study were devoid of both tdh and trh (Table 4).Despite this, there are studies suggesting that tdh/trh-negative strains can still cause human infection [9,40].This implies that even these 'non-pathogenic' strains should be considered in the realm of seafood safety monitoring.Several DNA markers, such as toxRS/new, orf8, HU-α, and PGS sequence, have been utilized to differentiate strains that belong to the 'pandemic group' [10][11][12][13].The findings of this study indicate that 45.8% of the isolates contained the PGS sequence (Table 4), which suggests that this marker may not be reliable for identifying the 'pandemic group', considering all strains in this study were non-pathogenic.Furthermore, none of the isolates were found to carry vopC and VPA1376, which are located within the Vp-PAI gene cluster (T3SS2), while 98.3% of the isolates were positive for vopQ, located in the T3SS1 gene cluster (Table 4).Additionally, all isolates were found to carry vopA2, which is part of the T6SS2 gene cluster, whereas only 39.0% of the isolates contained VP1409, part of the T6SS1 gene cluster (Table 4).These results align with the understanding that the T3SS1 and T6SS2 gene clusters are ubiquitous in all V. parahaemolyticus isolates, while the T3SS2 and T6SS1 gene clusters are more commonly identified in clinical isolates [41][42][43].All 59 isolates of V. parahaemolyticus exhibited swimming and swarming motility, as depicted in Fig 2 .These motilities are driven by polar and lateral flagella, respectively, and are crucial for the biofilm formation, especially in the initial stage, of V. parahaemolyticus [44].Notably, all isolates were capable of forming biofilms, with the majority being weak producers at 4˚C and moderate to strong producers at 25˚C or 37˚C (Fig 1).The capacity for biofilm formation at lower temperatures, such as 4˚C, may be significant for the bacteria's ability to persist on seafood surfaces over extended periods [18].The effect of culture temperature on V. parahaemolyticus biofilm formation was observed, with a markedly higher biofilm-forming ability at 25˚C compared to 15˚C and 37˚C [45].However, another study identified 37˚C as the optimal temperature for biofilm formation by this bacterium [46].The data presented here indicate that while the number of strong biofilm producers was similar at both at 25˚C and 37˚C, the biofilms produced at 25˚C were significantly stronger than those at 37˚C (Fig 1B).These results indicate that variables such as strain differences, culture media, and the type of culture containers can significantly influence the outcomes of biofilm-related phenotypes.Bacterial biofilms pose significant challenges to food safety in the food industry [47].Conventional approaches are insufficient to completely remove biofilms formed by Vibrionaceae family.However, advancements have been made with the development of safe and effective anti-biofilm strategies [48][49][50].For instance, natural compounds such as eugenol [51], carvacrol [52], quercetin [53], as well as essential oils [54,55], have demonstrated the ability to inhibit biofilm formation.Additionally, physical treatments like ultraviolet [56] and enzymatic treatments with flavourzyme [57] have shown promise in controlling biofilms in food products.Furthermore, the use of bacteriophages [58], lactic acid bacteria [59], and the manipulation of environmental conditions, such as altering glucose concentration [60,61], have been found to impact the growth of foodborne pathogenic biofilms on food surfaces.These approaches offer potential strategies for combating pathogenic microorganisms in the seafood industry, providing a means to enhance food safety.
The majority of the V. parahaemolyticus strains examined in this study exhibited high resistance to ampicillin, while displaying intermediate resistance to cefuroxime and cefazolin (Table 5).In contrast, clinical isolates from the stool samples of diarrhea patients in Nantong during 2018-2020 showed 68.2% and 75.6% high and moderate resistance to cefuroxime and cefazolin, respectively [9].This may indicate a significant divergence in the antibiotic resistance patterns between pathogenic and non-pathogenic V. parahaemolyticus strains in Nantong city.The common resistance to ampicillin among V. parahaemolyticus isolates is attributed to the presence of bla CARB-17 in all tested strains.This gene encodes a novel member of the CARB-17 family of β-lactamases, which confers intrinsic resistance to penicillins [62].Furthermore, V. parahaemolyticus strains isolated from ready-to-eat foods in China have been found to carry class 1 integrons with genetic structures such as dfrA14-bla VEB-1 -aadB and bla-VEB-1 -aadB-arr2-cmlA-bla OXA-10 -aadA1, which are associated with resistance to a range of antibiotics, including ampicillin [17].Nevertheless, additional research is warranted to explore the prevalence of antibiotic resistance genes among the V. parahaemolyticus strains identified in this study.This will provide a more comprehensive understanding of the resistance mechanisms in V. parahaemolyticus.
In summary, this study conducted an assessment of Vibrionaceae family in retail seafood products available in the Qidong market during the summer of 2023.The findings revealed that P. damselae and V. parahaemolyticus were the predominant species, with prevalence rates of 20.3% and 8.2%, respectively.Among the 59 V. parahaemolyticus strains isolated, none were pathogenic.These V. parahaemolyticus strains demonstrated proficiency in swimming and swarming behaviors, and a notable capacity to form biofilms.Furthermore, the study identified that the majority of V. parahaemolyticus isolates exhibited high resistance to ampicillin, intermediate resistance to cefuroxime and cefazolin, and sensitivity to other antibiotics tested.
The data presented here would provide theoretical guidance for food safety, and may be beneficial for controlling and treating seafood-related illnesses caused by Vibrionaceae family in Qidong City. ://doi.org/10.1371/journal.pone.0309304.t001

Fig 1 .
Fig 1. Biofilm formation by the V. parahaemolyticus isolates.A) Statistics on the number of biofilm producers.The number at the top of each bar represents the count of biofilm producers.B) The results of crystal violet staining assays.The asterisk (*) indicates a statistically significant difference (P < 0.01).https://doi.org/10.1371/journal.pone.0309304.g001

Fig 2 .
Fig 2. The swimming and swarming motility of the V. parahaemolyticus isolates.Statistics on the number of swimmers (A) and swarmers (C) and the results of swimming (B) and swarming motility (D).The number at the top of each bar indicates the count of swimmers or swarmers.The asterisks (*) indicate significant differences compared to V. parahaemolyticus RIMD2210633 (P < 0.01).The 'ns' symbols represent no significant differences (P > 0.01).https://doi.org/10.1371/journal.pone.0309304.g002

Table 4
, all isolates contained the gene tlh and toxR, but none were positive for tdh or trh.Furthermore, none of the isolates carried the genes toxR/ new, HU-α, ure, Mtase, vopC and VPA1376, with the exception of one isolate that possessed the orf8 gene.Additionally, 45.8% (27/59) of the isolates carried the PGS sequence, 98.3% (58/ 59) carried vopQ, 39.0% (23/59) carried VP1409, and 100% (59/59) carried vipA2.In summary, these findings suggest that the majority of the isolates were non-pathogenic, although the presence of certain virulence genes indicates that a small proportion may still have pathogenic potential.